Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry.
Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry.
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通过串联质谱法鉴定牛中分子量和低分子量神经丝蛋白的内源磷酸化位点。
DOI：
10.1021/bi030196q
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Deinzer,MaxL
中科院分区：
文献类型：
--
作者：
Trimpin,Sarah;Mixon,AprilE;Stapels,MarthaD;Kim,Moo-Young;Spencer,PeterS;Deinzer,MaxL
神经丝蛋白（NFP）是神经元细胞骨架中的中间丝。它们是高度磷酸化的，这种情况被认为是负责细丝的组装和稳定性。基质辅助激光解吸/电离（MALDI）飞行时间（TOF）质谱（MS）显示，牛NFP亚基的NFL、NFM和NFH分子量分别为63、105和125 kDa。质谱从头测序用于确定牛NFM的N-末端序列（115个氨基酸），这是以前未知的。分子量信息表明，NFL上有二分之一当量的磷酸基团，NFM上有24个。首次证明牛NFL有三个磷酸化位点（Ser 55，Ser 66和Ser 472）和NFM有22个（Ser512，Ser546，Ser554，Ser560，Thr627，Ser629，Ser634，Ser639，Thr646，Ser649，Ser654，Ser664，Ser669，Thr676，Ser679，Ser684，Ser694，Ser726，Ser750，Ser 756、Ser 770和Ser 846）和两个暂定位点（Ser 659/Thr 661和Thr 840）。Ser 66在其他物种的NFL中未被磷酸化，而两个位点（Ser 55和Ser 472）与在其他哺乳动物NFL中观察到的磷酸化一致。三个位点，Ser 55，Ser 66，Ser 472，是不均匀磷酸化的。牛NFM中的磷酸化主要发生在Lys-Ser-Pro（KSP）区域，但Val-Ser-Pro和Ser-Glu-Lys基序也被磷酸化。大多数的磷酸化位点与先前在其他哺乳动物NFM中鉴定的那些一致。在牛NFM中，22个位点中的16个总是磷酸化的（Ser 512、Thr 627、Ser 629、Ser 634、Ser 639、Thr 646、Ser 649、Ser 654、Ser 664、Ser 669、Thr 676、Ser 679、Ser 684、Ser 694、Ser 726和Ser 750），所有这些都包含在KSP区域中，六个有时被磷酸化（Ser 546、Ser 554、Ser 560、Ser 756、Ser 770和Ser 846）。NFP具有其他修饰，包括脱酰胺、氧化和N-末端乙酰化。还发生焦谷氨酸形成。
Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser55, Ser66, and Ser472) and NFM has 22 (Ser512, Ser546, Ser554, Ser560, Thr627, Ser629, Ser634, Ser639, Thr646, Ser649, Ser654, Ser664, Ser669, Thr676, Ser679, Ser684, Ser694, Ser726, Ser750, Ser756, Ser770, and Ser846) and two tentative sites (Ser659/Thr661and Thr840). Ser66was previously not known to be phosphorylated in NFL of other species, while two sites (Ser55and Ser472) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser55, Ser66, Ser472, are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser512, Thr627, Ser629, Ser634, Ser639, Thr646, Ser649, Ser654, Ser664, Ser669, Thr676, Ser679, Ser684, Ser694, Ser726, and Ser750), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser546, Ser554, Ser560, Ser756, Ser770, and Ser846). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.